ARMITAGE, J

DUQUESNOY, R

GRIFFITH, B

KAUFMAN, C

KEENAN, R

KORMOS, R

STARZL, T

YOUSEM, S

ZEEVI, A

ZERBE, T

English

PubMed

Keenan, R

Kaufman, C

Zeevi, A

Yousem, S

Zerbe, T

Armitage, J

Kormos, R

Duquesnoy, R

Griffith, B

Starzl, T

Name not available

Phenotypic T-cell changes in thoracic transplant recipients immunosuppressed with FK 506

D-Scholarship@Pitt

Phenotypic T-Cell Changes in Thoracic Transplant Recipients Immunosuppressed With FK 506 R. Keenan, C. Kaufman, A. Zeevi, S. Yousem, T. Zerbe, J. Armitage, R. Kormos, R. Dvquesnoy, B. Griffith and T. Starzl ' THE accurate diagnosis of rejection following pulmo-nary or cardiac transplantation presently requires the histologic interpretation of transbronchial or endomyocar-dial biopsies, respectively. Because these procedures are invasive, efforts have recently been directed toward find-ing alternative methods of determining the presence of active rejection episodes. Using 2-color flow q10metry, we examined changes in T-lymphocyte populations in 3 pulmonary and 3 cardiac transplant recipients in whom FK 506 was the sole immu-nosuppressive agent used. These patients were chosen because cytolytic lymphocyte therapy was not used and so circulating lymphocyte counts remained consistent throughout the posttransplant period. This allowed early and sequential T-cell subset analysis and comparison with histologic data simultaneously obtained from biopsies. MATERIALS AND METHODS Three pUlmonary transplam recipients (2 double lung and I heart-lung) received FK 506 as the only maintenance immuno-suppressive agent in the posttransplant period. These patients were followed for 90 to 285 days during which time each patient underwent 4 to 6 bronchoscopies with bronchoalveolar lavage (BAL) and transbronchial biopsy (TBB). Three cardiac transplant recipients were also included. and they too. received only FK 506 for immunosuppression. Cardiac patients were followed for 48 to 120 days and each undeNent 4 to 7 endomyocardial biopsies (EM B). The diagnosis of rejection was based on accepted his-tologic criteria for cardiac and pulmonary transplantation. Treat-ment of rejection episodes consisted of alterations in the dosage of FK 506 andlor a single bolus injection of conicosteroids. Total cell counts and differentials were performed on BAL fluid and on peripheral blood (PBL) obtained at the time of lavage or endomyocardial biopsy. Cells (100 ilL PBL or 1 x I(}' BAL) were suspended in 100 JLL of phosphate-buffered !>aline with 0.11)( sodium azide and labeled with various combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PEHabeled monoclo-nal antibodies at 4°C for 20 minutes. Red blood cells were then lysed using FACSlyse solution (Becton-Dickinson. Mountain View, CAl for 6 minutes. All cell suspensions were then centri-fuged for 5 minutes at 500 rpm. Supernatants were discarded and the cells resuspended in 0.5 ml phosphate-buffered saline and 0.5 mL \% paraformaldehyde. The following antibody combinations were used: Leukogate. nonspecific JgG, T/B. CD4/C[)8, TINK. CD4IDR. CD8IDR, T/afJ. p,o. (All antibodies were obtained from Becton·Dickinson. Mountain View. CA except for 'YO ob-tained from T Cell Sciences. Cambridge. MAl. Flow cytometry (2-color) was performed on a FACScan <Becton-Dickinson. Mountain View, CAl equipped with a Conson 30 computer program. Student t tests for unpaired samples were used to analyze alterations in the various T-cell subsets during active rejection episodes as compared to normal or quiescent biopsies. There was no difference in values oborined when the diagnOSis was acute cellular rejection or active otiliterative bronchiOlitis so they were considered together as acti .. e rejection episodes. RESULTS In pulmonary transplant patients_ the proportions of CD8+DR + and CD4+DR + T cells Io!xpressed as a percent of total CD8 and CD4) obtained fr0m BAL were signifi-cantly higher (P = .0015 and P = .00.::3. respectively) when there was histologic evidence of reje-':lion than were values obtained when the TBB was negati .. ~ or showed resolving rejection (Fig I). The proportion of DR + cells did not exceed 34% for CD4 + DR + and 21 o/c f0r CD8 + DR + subsets when biopsies were negative or shov.-ed resolving rejection while respective values were all gre~r than 43% and 40% during active rejection. Two of these episodes were asso-ciated with active cytomegalovirus infections (pneumoni-tis). The proportions ofCD8+DR+ and CD4+DR+ T cells obtained on these occasions were intermediate within the group of rejection values. Changes in the peripheral blood were less striking with only the propo..""l'1ion ofCD8+DR + T lymphocytes exhibiting significant increases during peri-ods of active rejection (P = .038) when compared to negative biopsies or to pretransplanc values (Fig 2). The separation in individual values for tK~ CD8+DR+ subset was much less distinct in the PBL. 'PJe largest proportion of PBL CD8+DR + T lymphocytes was seen in the 2 recipients with CMY pneumonitis .:.oincident with the onset of active obliterative bronchiolr.is. In the cardiac recipients (Fig 3), sirnilar increases in the proportion of CD8+ DR + T lymph~1Kes from PBL were associated with moderate (grade 2-3) rejection episodes (P = .01) when compared to values obuined at the time of negative or mild (grade 0-1) rejectiOCl biopsies. As with pulmonary transplant recipients. no ~ificant changes in From the Departments of Surgery and Pathology. University of Pittsburgh. Pittsburgh. Pennsylvania. Supported by Grants HL36416 and AIZ>467 from the National Institutes of Health and by the Pathology Eo.Jcation and Research Foundation, University of Pittsburgh. RK was recipient of a Canadian Lung Association Postgradu-ate Research Fellowship. Address reprint requests to Dr T. StarzI, Division of Transplan-tation Surgery. Falk Clinic, University of Piftsburgh. Pittsburgh. PA 15261. © 1991 by Appleton & Lange 004 1-13451911$3.001 +0 1162 Transplantation Proceedings. Vol 23. No 1 (February).. 1991: pp 1162-1164 PHENOTYPIC T-CELL CHANGES 100 Negative Rejection 80 6 8 60l 6 iii CMV -c:: • - ** G) I 0 I * 8 CMV -'-CMV ... I -4 8 G) "Or 8 a.. I II " 2O! -----I-III • 0 1 Ca~-aB CDS-DB CD4-DB CDS-[)8 C04 COS C04 coa Ftg'. The proportion of each DRT T-cell subset obtained from BAL lymphocytes of pulmonary transplant recipients when trans-brOnChial biopsy was negative or showed active rejection. The IT'ean value is indicated by the straight line. The number above the s,rnbols refers to the number of individual value points within each group. CMV beside the symbol refers to a measurement taken when CMV pneumonitis was also present. • P = .0023. rejection vs negative for CD4 +DR+/CD4+; "P = .0015. rejection vs negative tor COS'DR+/COS+. the proportion of CD4" DR + T cells were found in the peripheral blood. No significant changes in the proportion or number of T B. CD4/CD8. or TINK were found in either pulmonary or cardiac transplant recipients. The majority ofT cells in PBL and BAL were af3 receptor positive with few express-ing the 'Yf> receptor. 100 Negative Rejection l1li CMV 80 L I 6 -i I l1li CMV c:: 60r G) ~ I * ~ I 40r I 6 6 l1li I I I 20L II I 8 -Ic .. v • 0 Ca~-aB CDS-DB CO.-DB CDS-DB C04 COS C04 COS FIg 2. The proportion of each OR" T -<:ell subset obtained from peripheral blood lymphocytes from pulmonary transplant recipi-ents when transbronchiaJ biopsy was negative or showed active rejection. Refer to Fig 1 for explanation of straight line and CMV .• P = .038, rejection vs negative for COS+DR-.c08+. 1163 100 Negative Rejection 80 4 l1li -c:: 60 Q) 0 l1li ... , , Q) a.. 40 iii * 4 II ' , 20 II 4-III • B 0 II Ca~-aB CDS-DB IIil CD"-DR COS-DB C04 coa C04 COS Fig 3. The proportion of each DR + T -cell subset obtained from peripheral blood lymphocytes of heart transplant recipients when endomyocardial biopsy was negative or showed active rejection. The mean value is indicated by the straight line .• P = .01. rejection vs negative for COS'OR+/COS-. DISCUSSION AND CONCLUSIONS These 'results suggest that graft-infiltrating T lymphocytes (BAL) exhibit a greater degree of activation during pulmo-nary rejection as measured by significant increases in the proportions of both CD4 +DR + and CDS' DR + T-cell pop-ulations whereas lymphocytes recovered from peripheral blood express only CD8+DR+ activity. This also suggests that local events within the graft. particularly activation of the CD4 + DR + subset. are not fully reflected by changes seen in the peripheral blood. This is also the first report. in patients immunosuppressed with FK 506. to document an increase in DR - T cells with active rejection in pulmonary and cardiac transplant recipients. In renal transplantation. previous investigators using T lymphoc}1eS obtained from fresh kidney biopsies. have also documented an associa-tion between CD3+DR+ or CD8+DR' subsets and the presence of acti·.e rejection 1.2 but this is the first study to demonstrate that activated CD4 + cells also accumulate during rejection episodes. Cytomegalovirus infections are also known to cause lymphocyte activation with expression of the DR+ marker resulting in nonspecific elevation of the CD8 + DR + T -ceil subset in the peripheral blood of renal~ transplant recipi, ents during both rejection and infection. Similar increases in PBL CDS + DR + T cells during infection were also seen in cardiac transplant recipients"·5 although the association with rejection was less clear. In this study. 2 episodes of active obliterative bronchiolitis, accompanied by CMV pneumonitis. were associated with the largest increases in PBL CDS +DR + T cells. Of interest is that values obtainerl from BAL lymphocytes. at the site of infection. were only intermediary within the range of values from rejection episodes. The proportion of CD4 + DR + T lymphocytes in either PBL or BAL did not appear to be affected by the presence of CMV. These studies support our observation 1164 that use of peripheral blood T lymphocytes may not be sufficient to distinguish rejection from CMY infection and that analysis of graft-infiltrating cells more accurately reflects intragraft events. The results from this study suggest that analysis of DR + subsets of T cells, particularly CD8 + DR +, may be helpful in the routine monitoring of thoracic transplant recipients in that significant changes occur coincident with the his-tologic diagnosis of active rejection. Analysis of subpop-ulations of BAL lymphoc~les shows that proportions of DR + cells greater than 4()q are always associated with active rejection whether acute or chronic. Elevations in the CD8 + DR + subset of PBL lymphocytes indicates a probability of active rejectiun or CMY infection which, in pulmonary transplant recipients, would lead to further KEENAN, KAUFMAN, ZEEVI ET AI. testing to obtain a diagnosis. Whether these tests will be . useful as a screening tool for rejection is the subject of ongoing investigation. REFERENCES I. Gross U, Cunningham P. Sash C: Transplant Proc 20:102 1~U ' 2. Totlerman TH. Hanas E, Bergstrom R: Transplantation 47:817.1989 3. van Es A. Baldwin WM, Oljans Pl: Transplantation 37:65, 1~4 4. Roodman ST. !>1iller LW. Tsai CC: Transplantation 45: 1050 19~ , 5. Fieguth HG. Wahlers T. Dammenhayn L: J Heart Trans. plant 8:87. 1989 

PHENOTYPIC T-CELL CHANGES IN THORACIC TRANSPLANT RECIPIENTS IMMUNOSUPPRESSED WITH FK506

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PHENOTYPIC T-CELL CHANGES IN THORACIC TRANSPLANT RECIPIENTS IMMUNOSUPPRESSED WITH FK506

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